We seek to develop a novel, direct, and generally applicable selection strategy for identifying enzymatic activity and specificity towards small-molecule, drug-like compounds. Both site-directed mutagenesis and combinatorial protein engineering (directed evolution) have successfully improved biocatalysts, however, full utilization of directed evolution is hampered by the lack of a general selection strategy to identify variants with improved properties. Our method comprises two steps: i) a nuclear receptor is engineered to activate transcription in response to a small-molecule reaction product, coupling the presence of the small molecule to survival of a microbe (i.e. genetic selection); ii) an enzyme is engineered through genetic selection to catalyze formation of the desired product. While applicable to any small molecule-enzyme pair, in this project we develop the method on the enzyme-catalyzed synthesis of beta-lactam antibiotics. This proposal seeks to improve ligand-activated growth (Aim 1), to develop enzyme-activated growth (Aim 2), to enhance enzyme substrate specificity to include beta-lactamase-resistant structures (Aim 3), and to synthesize biocatalytically a synthetic antibiotic currently not accessible through enzymatic synthesis (Aim 4). Our long-range goal is the application of this method to any drug-like target molecule, including non-lactam-based antibiotics and other drug classes.